Identification Of Cis-element And Trans-factors Functioning In Nuclear Retention Of LncRNA MEG3

Research BackgroundThe advent of next generation sequencing data reveal that most of the human genome is transcribed Among these transcripts,Long Noncoding RNAs(lncRNAs)are the most abundant class of RNAs that play quite diverse roles and multiple functions by interacting with RNAs,DNA and proteins Similar to messenger RNAs(mRNAs),lncRNAs are transcribed by RNA polymerase II(pol II),capped at 5' end,most of them have splicing events and Polyadenylate(polyA)tail.According to definition,lncRNAs do not encode proteins,which is the fundamental difference between lncRNAs and mRNAs.For the localization,all mRNAs are exported to the cytoplasm for translation,whereas lncRNAs could be in the nucleus,cytoplasm or in both compartments.The efficient export of spliced mRNAs to the cytoplasm is apparently due to recruitment of TRanscription-EXport(TREX)complex to 5' end of mRNA during splicing,in contrast,many spliced lncRNAs are retained in the nucleus despite of splicing events.Several studies have already provided some clues on nuclear retention of lncRNAs.For example,lncRNA FIRRE contains 156 bp repeating RNA domain that accumulate FIRRE on its target chromosome.Similarly,XIST 5' repeat element helps XIST RNA to localize on X chromosome.In both cases,depletion of hnrmpU results in the delocalization of RNAs from their target sites on the chromosomes.Another study suggests Malatl contains two independent regions(each of lkb)those facilitate the localization of Malatl to nuclear speckles.Knockdown of nuclear speckle enriched proteins RNPS1,SRm160 and IBP160 showed diffused malatl signal in the nucleoplasm.Recently,a short pentamer RNA motif(AGCCC)exist in mouse IncRNA(BORG)is shown to be responsible for its nuclear retention.However,detailed mechanisms for nuclear retention of spliced lncRNAs are still elusiveResearch MethodsIn Maternally Expressed Gene 3(MEG3)reporter,beta globin(?-globin)intron was inserted upstream of MEG3 complementary DNA(cDNA)to create a splicing event.MEG3 truncation and deletion constructs were designed to map MEG3 Nuclear Retention Element(NRE).For evaluating NRE potential,chimeric constructs of NRE-ANCR and NRE-?-globin Wild Type(WT)were designed.Reporter's expression and localization was analyzed by Reverse Transcriptase Polymerase Chain Reaction(RT-PCR)and Fluorescence In Situ Hybridization(FISH),respectively.To find NRE interacting protein we used biotin-streptavidin affinity purification followed by Mass Spectrometry(MS)analysis.We screened the MS proteins list using Small Interfering RNAs(siRNAs)and checked its effect on MEG3 localization.To get the global effect of U1 small nuclear ribonucleoprotein(U1 snRNP)components on the distribution of spliced lncRNAs we depleted U1 snRNP components and isolated RNA from nuclear and cytoplasmic fractions for RNA-seq analysisResultsTo gain deep insight into the mechanism of how some spliced IncRNAs are retained in the nucleus,we constructed MEG3 lncRNA reporter,MEG3 cDNA was cloned to a vector downstream of an optimized ?-globin intron 1.To determine the localization of the reporter transcripts,these reporters were transiently transfected into HeLa cells,RT-PCR was then performed on RNAs purified from the cytoplasmic and nuclear compartments.The cytoplasm/nucleus separation efficiency was checked using western blot of nuclear marker UAP56 and cytoplasmic marker tubulin.Consistent with previous studies,MEG3 was mainly detected in the nucleus,whereas ANCR was detected both in the cytoplasm and the nucleus.The localization of these transcripts were further assayed using RNA-FISH,MEG3 reporter transcripts were observed dominantly in the nucleus,compared to a more even distribution in the cytoplasm and nucleus for Anti-differentiation Noncoding RNA(ANCR)reporter transcripts.Together,these data suggested the MEG3 reporter we constructed could be used for mechanistic analysis of nuclear retention for spliced IncRNAHypotheses of spliced lncRNA retained in the nucleus may include failure to recruit export machinery or retained by specific retention mechanism.To test the first hypothesis,we purified MEG3 Ribonucleoprotein(RNP)assembled in vivo and performed mass spectrometry analysis,most of the TREX components were identified such as THOC2,THOC1,THOC3,THOC5,THOC6,ALYREF and UAP56,which suggested the retention of MEG3 in the nucleus is unlikely due to the failure to recruit export machinery.Next we further tested the hypothesis that MEG3 may contain nuclear retention element.A series deletion constructs were made to truncate MEG3 from the 3' end,transfection of these constructs to HeLa cells followed by RNA-FISH revealed that removal of the 3 end up to 413 nt(nucleotides)had no significant impact on the nuclear localization of MEG3 transcripts,further truncations resulted in partial export of MEG3 transcripts until the truncation of 769 nt,which led to striking cytoplasmic localization of MEG3 transcript.Consistently,removal of the 5' of MEG3(1-797 nt)showed no impact to the localization of MEG3,further mapping revealed that a 356nt sequence(from 798 to 1153 nt)served as nuclear retention element(NRE),the transcripts from this construct were typically retained in the nucleus,deletion of the NRE led to complete cytoplasmic localization,and partial deletion of NRE resulted in both cytoplasmic and nuclear localization of MEG3 transcripts.Thus we concluded that the nuclear localization of spliced lncRNA MEG3 was due to the presence of NRE in the transcriptsTo test whether the MEG3 NRE functions in heterogeneous context,we inserted it at the 5' of ANCR or ?-globin wild-type constructs.RT-PCR revealed that insertion of the NRE had no impact on the splicing of either ANCR or ?-globin WT transcripts.However,by RNA-FISH,insertion of the NRE to ANCR led to almost complete retention of ANCR transcripts to the nucleus and partial but apparent nuclear retention of spliced ?-globin transcripts,suggesting MEG3 NRE is potent to retain not only spliced IncRNA but also spliced mRNA in the nucleusTo investigate which trans-factors bind to the MEG3 NRE and mediate the nuclear retention,we first purified RNP assembled in vitro on MEG3 NRE using biotin-streptavidin strategy,NRE transcripts without biotin labeling and the antisense of NRE were used as negative controls.RNP assembled on NRE showed more protein enrichment after separation on Polyacrylamide Gel Electrophoresis(PAGE)as compared to RNPs assembled on NRE without biotin labeling or on the antisense NRE Mass spectrometry analysis revealed major protein components in each RNP.Western-blot confirmed several proteins enriched in RNP assembled on MEG3 NREScreen of the components identified in MEG3 NRE RNP using siRNA revealed that depletion of Small Nuclear Ribonucleoprotein Polypeptide A(SNRPA)led to cytoplasmic localization of MEG3 in about 50%of cells transfected with MEG3 construct.As SNRPA is a component of U1 snRNP,we further screened other components in U1 snRNP.Depletion of Small Nuclear Ribonucleoprotein 70(SNRNP70)or Small Nuclear Ribonucleoprotein Polypeptide D2(SNRPD2)resulted in cytoplasmic localization of MEG3 in 45%or 70%of transfected cells individually.Consistent with observation in RNA-FISH,RT-PCR also indicated significant increase of MEG3 transcripts in the cytoplasm after depletion of SNRPA,SNRNP70 or SNRPD2.Further FISH-Immunofluorescence(FISH-IF)confirmed that cytoplasmic signal of MEG3 reporter's expression is due to the Knockdown(KD)of SNRPA,SNRNP70 or SNRPD2To investigate whether these factors affect the localization of endogenous lncRNAs,we simultaneously depleted all three proteins in HFF1 cells,and the RNA was purified for RNA sequencing from cytoplasmic or nuclear compartments.RNA Sequencing(RNA-seq)data revealed that more lncRNAs were detected in the cytoplasm.Specifically using RT-PCR,for endogenous MEG3,we observed 44%in the cytoplasm after depletion as compared to 26%in the control Similarly,for the other three lncRNAs with dominant nuclear localization,depletion of SNRPA,SNRNP70 and SNRPD2 led to an increase of 33%to 78%(PVT1),25%to 52%(TUG1)and 20%to 41%(PSMA3-AS1)in the cytoplasm.These data together supported that U1 snRNP components play crucial role for restraining spliced lncRNAs in the nucleusConclusionIn summary,lncRNA MEG3 has a potent nuclear retention element in the middle of MEG3 RNA sequence,the NRE functions both in its natural and heterologous context.This NRE recruited U1 snRNP components to restrain the spliced lncRNAs in the nucleus.Our data further support cis element as determinant of lncRNA localization and the importance of U1 snRNP components in subcellular distribution of spliced lncRNAs.